KR102608336B1 - Novel peptide derived from pep27 peptide and uses thereof - Google Patents

Abstract

The present invention relates to a new peptide, and more specifically, in the amino acid sequence of SEQ ID NO: 1: i) the 2nd and 4th amino acids are substituted with tryptophane (W); ii) the 2nd, 4th, 11th and 13th amino acids are replaced with tryptophan; iii) Peptides in which the 2nd, 4th, 19th, 20th, 22nd, 23rd, 26th, and 27th amino acids are substituted with tryptophan exhibit excellent antibacterial activity and low cytotoxicity, and are ultimately used in antibiotics, cosmetic compositions, It can be usefully used as an active ingredient in food additives, feed additives, biological pesticides, and quasi-drugs.

Description

Novel peptides derived from PEP27 peptide and uses thereof {NOVEL PEPTIDE DERIVED FROM PEP27 PEPTIDE AND USES THEREOF}

The present invention relates to novel peptides derived from PEP27 peptide and their uses.

Bacterial infections are one of the most common and fatal causes of human disease, and unfortunately, the overuse of antibiotics has led to antibiotic resistance in bacteria. In fact, the rate at which bacteria develop resistance to new antibiotics occurs much faster than the rate at which analogues of new antibiotics are developed. For example, life-threatening bacterial species such as Enterococcus faecalis , Mycobacterium tuberculosis , and Pseudomonas aeruginosa are resistant to all known antibiotics. has developed resistance to

Antibiotic resistance, a phenomenon distinct from resistance to antibiotics, was first discovered in Pneumococcus sp. in the 1970s and provided important clues to the mechanism of action of penicillin. Resistant species stop growing but do not die in the presence of conventional concentrations of antibiotics. Resistance occurs because when antibiotics inhibit cell wall synthesis enzymes, the activity of bacterial autolytic enzymes such as autolysin does not occur. This fact occurs because penicillin activates endogenous hydrolytic enzymes. This kills bacteria and inhibits their activity, resulting in their survival even when treated with antibiotics. It is clinically very important for bacteria to be resistant to various antibiotics, because if it becomes impossible to eradicate resistant bacteria, the effectiveness of antibiotic treatment in clinical infections decreases. In addition, the development of resistance is considered a prerequisite for the development of resistance to antibiotics because it creates strains that survive despite antibiotic treatment. These strains acquire new genetic elements that make them resistant to antibiotics and continue to grow even in the presence of antibiotics. In fact, it is known that all resistant bacteria are also resistant, so it is necessary to develop new antibiotics that can kill these antibiotic-resistant bacteria. In terms of the mechanism of action, antibiotic resistance is largely composed of two pathways. The first is phenotypic resistance that occurs when the growth rate is reduced in all bacteria, and the second is genetic resistance caused by mutations that occur in specific bacteria. am. In both cases, the basic phenomenon is that downregulation of autolysin activity occurs. This downregulation is temporary in the case of apparent resistance to external stimuli and causes changes in the pathways that control cell hemolysis. In the case of genetic resistance caused by mutation, it is permanent. The simplest case of genetic resistance is a defect in the autolysin enzyme. However, for various reasons that are unclear, no strain with resistance due to a defect in this suicide enzyme has been clinically discovered. Rather, clinical resistance is caused by a defect in the autolysin enzyme. This is achieved by regulating the activity of lysine. In order to combat bacteria that are resistant to antibiotics, the development of new antibiotics is necessary, and in addition, the development of new antibiotics that act independently of autolysin activity is necessary.

Meanwhile, bacteriocin is a natural antibacterial protein produced by various types of microorganisms and has a bactericidal effect on bacterial species similar to the producing bacteria. Bacteriocins are structurally classified into three classes. The first are lantibiotics, the second are nonlantibiotics, and the third are those secreted by signal peptides. Animals, including insects, also produce naturally occurring peptide antibiotics, which are structurally divided into three groups. The first is a cysteine-rich β-sheet peptide, the second is an α-helical amphiphilic molecule, and the third is a proline-rich peptide. These antibacterial peptides are known to play an important role in host defense and the innate immune system, and these antibacterial peptides have various structures depending on their amino acid sequences.

Korean Patent No. 2158036

The purpose of the present invention is to provide new peptides and uses thereof.

1. In the amino acid sequence of SEQ ID NO: 1, i) the 2nd and 4th amino acids are replaced with tryptophane (W); ii) the 2nd, 4th, 11th and 13th amino acids are substituted with tryptophan; iii) A peptide in which the 2nd, 4th, 19th, 20th, 22nd, 23rd, 26th and 27th amino acids are substituted with tryptophan.

2. In item 1 above, the peptide has the amino acid sequence of SEQ ID NO: 3.

3. The peptide of 1 above, wherein the peptide has antibacterial activity against at least one selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant bacteria.

4. The peptide of 3 above, wherein the Gram-positive bacteria are at least one selected from the group consisting of Staphylococcus aureus, Bacillus subtilis , and Listeria monocytogenes .

5. The peptide of 3 above, wherein the Gram-negative bacteria are at least one selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa , and Salmonella typhimurium .

6. In 3 above, the antibiotic-resistant bacteria are antibiotic-resistant Staphylococcus aureus ( S. aureus ), Escherichia coli ( E. coli ), Pseudomonas aeruginosa ( P. aeruginosa ), and Salmonella Typhimu. At least one peptide selected from the group consisting of Salmonella typhimurium .

7. An antibacterial composition containing the peptide of any one of items 1 to 6 above as an active ingredient.

8. The antibacterial composition of item 7 above, wherein the antibacterial composition is a pharmaceutical composition.

9. The antibacterial composition of item 7 above, wherein the antibacterial composition is any one selected from the group consisting of cosmetic compositions, food additives, feed additives, biological pesticides, preservative compositions, and quasi-drug compositions.

The peptide of the present invention exhibits excellent antibacterial activity and low cytotoxicity, and can therefore be usefully used as an active ingredient in antibiotics, cosmetic compositions, food additives, feed additives, biological pesticides, and quasi-drugs.

Figure 1 shows the results of confirming the secondary structure of the antibacterial peptide PEP27 (control group) and the new peptide PEP27-2 (experimental group), a PEP27 analog with substituted amino acid residues, in a membrane-like environment. ), Figure 1b refers to a bacterial membrane-mimicking artificial membrane composed of PE/PG (7/3), and Figure 1c refers to a eukaryotic cell membrane-mimicking artificial membrane composed of PC/CH/SM (1/1/1).
Figure 2 shows the results of confirming the action of the control PEP27 peptide and the new peptide PEP27-2 on the Escherichia coli membrane using flow cytometry (FACS).
Figure 3 shows the results of confirming the binding ability of the control PEP27 peptide and the new peptide PEP27-2 to DNA, an internal material of bacteria. The peptide:DNA ratios are DNA alone (0), 0.25:1, 0.5:1, and 1:1, respectively. , 1.5:1, 2:1, 3:1, 4:1.
Figure 4 shows the results of confirming the action of the control PEP27 peptide and the new peptide PEP27-2 on the membranes of E. coli and Staphylococcus aureus using scanning electron microscopy (SEM).
Figure 5 shows the results of fluorescence microscopy confirming the internalization of the FITC-labeled PEP27-2 peptide (FITC-PEP27-2) in a human keratinocyte cell line (HaCaT cell line, Dr. NE. Fusenig, Heidelberg, Germany).
Figure 6 shows the results of confirming the healing effect of PEP27 peptide and the antibiotic ceftriaxone on abscesses formed after infection with the multi-drug resistant strain Starphylococcus aureus MW2, when treated alone or in combination.
Figure 7 shows the results of confirming the total number of bacteria recovered (CFU/g) by recovering the abscess area formed after infection with Staphylococcus aureus MW2, a multi-drug resistant strain.
Figure 8 shows PEP27 peptide and the antibiotic ceftriaxone for abscesses formed after infection with a strain (Staphylococcus aureus MW2-GFP) in which a GFP (green fluorescent protein) gene was inserted into the multidrug-resistant strain Staphylococcus aureus MW2. This is the result of confirming the distribution of strains and the healing effect when treated alone or in combination with (ceftriaxone).
Figure 9 shows the results of confirming changes in abscess tissue when the abscess formed after infection with the multi-drug resistant strain Staphylococcus aureus MW2 was treated alone or in combination with PEP27 peptide and the antibiotic ceftriaxone.
Figure 10 shows the results of confirming the relative expression rates of genes related to the inflammatory response in abscess tissue formed after infection with Staphylococcus aureus MW2, a multidrug-resistant strain (TNF-α: tumor necrosis factor-alpha, IL-1β: interleukin- 1 beta, IL-6: interleukin-6, iNOS: nitric oxide synthase, COX-2: cyclooxygenase-2).

Hereinafter, the present invention will be described in detail.

The present invention relates to the amino acid sequence of SEQ ID NO: 1 where i) the 2nd and 4th amino acids are substituted with tryptophane (W); ii) the 2nd, 4th, 11th and 13th amino acids are replaced with tryptophan; iii) Provides a peptide in which the 2nd, 4th, 19th, 20th, 22nd, 23rd, 26th and 27th amino acids are substituted with tryptophan.

PEP27, the parent peptide with the previously known amino acid sequence of SEQ ID NO: 1, is secreted by Streptococcus pneumoniae , and PEP27 is known as a "death signal peptide" present in the Vex123-Pep27-VncRS locus.

[SEQ ID NO: 1]

MRKEFHNVLSSGQLLADKRPARDYNRK-NH ₂

PEP27 peptide can be produced by conventional peptide synthesis methods known in the art, and the production method is not particularly limited. For example, as a method for synthesizing the PEP27 peptide, conventional peptide chemical synthesis methods in the art can be used, specifically solution-phase peptide synthesis and solid-phase peptide synthesis. , fragment condensation method, and F-moc or T-BOC chemical methods can be used.

The peptide of the present invention may be a peptide having the amino acid sequence of SEQ ID NO: 2 to SEQ ID NO: 4.

[SEQ ID NO: 2]

MWKWFHNVLSSGQLLADKRPARDYNRK-NH ₂

[SEQ ID NO: 3]

MWKWFHNVLSWGWLLADKRPARDYNRK-NH ₂

[SEQ ID NO: 4]

MWKWFHNVLSSGQLLADKWWAWWYNWW-NH ₂

The peptide having the amino acid sequence of SEQ ID NO: 2 was a peptide in which the 2nd and 4th amino acids were substituted with tryptophan (W) from the parent peptide, PEP27, and was named PEP27-1.

The peptide having the amino acid sequence of SEQ ID NO: 3 was a peptide in which the 2nd, 4th, 11th, and 13th amino acids from the parent peptide, PEP27, were substituted with tryptophan (W), and was named PEP27-2.

The peptide having the amino acid sequence of SEQ ID NO: 4 is a peptide in which the 2nd, 4th, 19th, 20th, 22nd, 23rd, 26th, and 27th amino acids from the parent peptide, PEP27, are substituted with tryptophan (W), It was named PEP27-5.

“-NH ₂ ” in the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4 indicates that the carboxyl group at the C terminus is amidated.

Specifically, the peptide of the present invention may be a peptide having the amino acid sequence of SEQ ID NO: 3.

By substitution of the above-mentioned amino acids, the electrode of the peptide can be increased/decreased and cytotoxicity can be reduced. In addition, by substitution of the above-mentioned amino acids, the peptide can exhibit antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant bacteria.

The peptide of the present invention can exhibit antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant bacteria.

The gram-positive bacteria may be at least one selected from the group consisting of Staphylococcus , Listeria , Corynebacterium , Lactobacillus , and Bacillus . Not limited. Specifically, the peptide of the present invention exhibits excellent antibacterial activity against at least one selected from the group consisting of Staphylococcus genus, Bacillus genus, or Listeria genus.

The gram-negative bacteria may be at least one selected from the group consisting of Pseudomonas , Escherichia , Salmonella , Leptospira , and Rickettsia , but is not limited thereto. Specifically, the peptide of the present invention exhibits excellent antibacterial activity against at least one selected from the group consisting of Pseudomonas genus, Escherichia genus, or Salmonella genus.

Antibiotic-resistant bacteria are at least selected from the group consisting of antibiotic-resistant Pseudomonas aeruginosa , Escherichia coli, Salmonella typhimurium , and Staphylococcus aureus . It may be one, but is not limited thereto. The above antibiotics include aminoglycoside series (aminoglycoside, gentamicin, neomycin, etc.), penicillin series (ampicillin, etc.), sulfonamide series, beta-lactam series (beta-lactam, amoxicillin/clavulanic acid, etc.), and chloramphenicol. A group consisting of antibiotics of the erythromycin series, florfenicol series, fosfomycin series, kanamycin series, lincomycin series, methicillin series, quinolone series, streptomycin series, tetracycline series, trimethoprim series, and vancomycin series. It may be at least one selected from, but is not limited to.

The peptide of the present invention exhibits excellent antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant bacteria, while exhibiting low cytotoxicity against human-derived cells within the antibacterial concentration.

Additionally, the present invention provides an antibacterial composition containing the above-described peptide as an active ingredient.

The antibacterial composition may be a pharmaceutical composition. That is, the present invention provides an antibacterial pharmaceutical composition containing the above-described peptide as an active ingredient.

SEQ ID NOs: 2 to 4, which are analogue peptides derived from the PEP27 antibacterial peptide of the present invention, exhibit strong antibacterial activity and low cytotoxicity to human-derived cells, so the peptide of the present invention is useful as an active ingredient in an antibacterial pharmaceutical composition. can be used For example, the peptides of the present invention can be used as antibiotics. For example, the peptide of the present invention can be used in combination with other antibiotics, and when used in combination with other antibiotics, it can exhibit synergistic antibacterial activity compared to when used alone.

In addition to the peptide as an active ingredient, the pharmaceutical composition may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.

Carriers, excipients, and diluents that may be included in pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, and methyl cellulose. , microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

Pharmaceutical compositions can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations. Parenteral administration may mean administration through routes of administration other than oral, such as rectal, intravenous, peritoneal, muscular, arterial, transdermal, nasal, inhalation, ocular, and subcutaneous.

The pharmaceutical composition may additionally contain one or more active ingredients that exhibit the same or similar functions as the peptide, which is the active ingredient.

For example, the pharmaceutical composition of the present invention may further include known antibiotics.

Known antibiotics include aminoglycoside series (aminoglycoside, gentamicin, neomycin, etc.), penicillin series (ampicillin, etc.), sulfonamide series, beta-lactam series (beta-lactam, amoxicillin/clavulanic acid, etc.), Consists of antibiotics of the chloramphenicol series, erythromycin series, florfenicol series, fosfomycin series, kanamycin series, lincomycin series, methicillin series, quinolone series, streptomycin series, tetracycline series, trimethoprim series, and vancomycin series. It may be at least one selected from the group. Specifically, the antibiotics are meropenem, ceftazidime, ceftriaxone, doripenem, ertapenem, imipenem, and cilastatin, cefadroxil, cefazolin, cephalexin, cefaclor, cefotetan, cefoxitin, and cefprozil. , cefuroxime, cefdinir, cefditoren, cefixime, cefotaxime, cefpodoxin, ceftibuten, cefepime, and ceftaroline. More specifically, the antibiotic may be at least one selected from the group consisting of meropenem, ceftazidime, and ceftriaxone.

The effective dose of the peptide of the present invention may be 0.1 to 2 mg/kg and may be administered once to three times a day.

The total effective amount of the peptide of the present invention can be administered to the patient in a single dose in the form of a bolus or by infusion for a relatively short period of time, and multiple doses can be administered for a long period of time. It may be administered by a fractionated treatment protocol. The concentration is determined by considering various factors such as the drug administration route and number of treatments as well as the patient's age and health condition, so that the effective dose for the patient is determined. Considering this, those skilled in the art will understand the present invention. It will be possible to determine an appropriate effective dosage according to the specific use of the peptide as an antibiotic.

The antibacterial composition may be any one selected from the group consisting of cosmetic compositions, food additives, feed additives, biological pesticides, preservative compositions, and quasi-drug compositions.

The present invention provides an antibacterial cosmetic composition containing the above-described peptide as an active ingredient.

SEQ ID NOs: 2 to 4, which are analogue peptides derived from the PEP27 antibacterial peptide of the present invention, exhibit strong antibacterial activity and low cytotoxicity to human-derived cells, so the peptide of the present invention is useful as an active ingredient in antibacterial cosmetic compositions. can be used

The cosmetic composition may contain ingredients commonly used in cosmetic compositions in addition to the peptide, which is an active ingredient. For example, the cosmetic composition may further include at least one of antioxidants, stabilizers, solubilizers, vitamins, conventional auxiliaries such as pigments and fragrances, and a carrier.

The cosmetic composition may contain 0.1 to 50% by weight of the peptide, which is an active ingredient, and preferably 1 to 10% by weight.

Cosmetic compositions can be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, It may be formulated as powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto.

When the cosmetic composition is in the form of a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacantha, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier ingredients. It can be.

When the formulation of the cosmetic composition is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient, and especially in the case of spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the cosmetic composition is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar or tragacantha can be used.

When the formulation of the cosmetic composition is a cleansing surfactant, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

The present invention provides an antibacterial food additive containing the above-mentioned peptide as an active ingredient.

SEQ ID NOs. 2 to 4, which are analogue peptides derived from the PEP27 antibacterial peptide of the present invention, exhibit strong antibacterial activity and low cytotoxicity to human-derived cells, so the peptide of the present invention is useful as an active ingredient in an antibacterial food additive. can be used

When using the peptide of the present invention as a food additive, the peptide can be added as is or used together with other food ingredients. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use. For example, the peptide of the present invention may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the food raw material. However, in case of long-term intake, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Foods to which the peptide of the present invention can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, etc. It may be an alcoholic beverage or a vitamin complex, and includes all foods in the conventional sense.

The present invention provides an antibacterial feed additive containing the above-mentioned peptide as an active ingredient.

SEQ ID NOs. 2 to 4, which are analogue peptides derived from the PEP27 antibacterial peptide of the present invention, exhibit strong antibacterial activity and low cytotoxicity to human-derived cells, so the peptide of the present invention is useful as an active ingredient in an antibacterial feed additive. can be used

When the peptide of the present invention is used as a feed additive, it replaces existing antibiotics and inhibits the growth of harmful food pathogens to improve the health of the animal, improve the weight gain and meat quality of livestock, and increase milk production and immunity. It can show an effect. When the peptide of the present invention is used as a feed additive, the feed can be manufactured in the form of fermented feed, compounded feed, pellet form, and silage.

Fermented feed can be manufactured by fermenting organic matter by adding various microorganisms or enzymes other than the peptide of the present invention, and compounded feed can be manufactured by mixing various types of general feed with the peptide of the present invention. Pellet-type feed can be manufactured by applying heat and pressure to the above-mentioned mixed feed, etc. in a pellet machine, and silage can be manufactured by fermenting green feed with microorganisms. Wet fermented feed collects and transports organic matter such as food waste, mixes excipients for sterilization and moisture control at a certain ratio, and then ferments for more than 24 hours at a temperature suitable for fermentation to ensure that the moisture content is approximately 70%. It can be manufactured by adjusting it. Fermented dried feed can be manufactured by adjusting wet fermented feed to an additional drying process so that the moisture content is about 30% to 40%.

The present invention provides an antibacterial preservative composition, an antibacterial biological pesticide, and an antibacterial quasi-drug containing the above-described peptide as an active ingredient.

SEQ ID NOs: 2 to 4, which are analogue peptides derived from the PEP27 antibacterial peptide of the present invention, exhibit strong antibacterial activity and low cytotoxicity to human-derived cells, so the peptide of the present invention can be used in antibacterial preservative compositions, antibacterial biological pesticides, And it can be usefully used as an active ingredient in antibacterial quasi-drugs.

Preservative compositions may include food preservatives, cosmetic preservatives, or pharmaceutical preservatives. Food preservatives, cosmetic preservatives, and pharmaceutical preservatives are additives used to prevent deterioration, decay, discoloration, and chemical changes. They may include disinfectants and antioxidants, and they inhibit the growth of microorganisms such as bacteria, mold, and yeast, thereby inhibiting the growth of microorganisms such as bacteria, mold, and yeast. In pharmaceuticals, functional antibiotics that inhibit the growth of putrefactive microorganisms or have a sterilizing effect may also be included. The ideal conditions for such a preservative composition are that it should be non-toxic and effective even in trace amounts.

When using the composition of the present invention as a quasi-drug composition, the peptide, which is an active ingredient, can be added as is or used together with other quasi-drugs or quasi-drug ingredients.

The quasi-drug composition may be a disinfectant cleaner, shower foam, garnish, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment, patch, or filter filler.

Additionally, the present invention provides an antibacterial method comprising administering a pharmaceutically effective amount of the antibacterial peptide to a subject.

The entity may be, but is not limited to, a mammal other than a human.

Hereinafter, the present invention will be described in detail with reference to examples. However, the examples below are provided only for illustrative purposes to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

Example

1. Synthesis and isolation purification of peptides

According to Merrifield's liquid peptide synthesis method (Merrifield, RB., J.Am. Chem. Soc., 85, 2149, 196), 2 in the amino acid sequence of PEP27, the parent peptide shown in the amino acid sequence of SEQ ID NO: 1. The peptide was synthesized (SEQ ID NO: 2) by substituting the 4th and 4th residues with tryptophan (W). A peptide was synthesized by substituting the 11th, 13th, 19th, 20th, 22nd, 23rd, 26th, and 27th residues in the amino acid sequence of SEQ ID NO: 2 with tryptophan (Table 1).

Specifically, Rink Amide MBHA-Resin was used as a starting material for the peptide designed in the present invention whose carboxyl terminus is in NH2 form, and for the peptide whose carboxyl terminus is in OH form, Fmoc (9 -fluorenylmethoxycarbonyl)-amino acid-Wang Resin was used as a starting material. Peptide chain extension by Fmoc-amino acid coupling was performed by the DCC (N-hydroxybenzo trizole (HOBt)-dicyclo-hexycarbodiimide) method. After coupling the Fmoc-amino acid at the amino terminus of each peptide, the Fmoc group was removed with NMP (20% piperidine/N-methyl pyrolidone) solution, washed several times with NMP and DCM (dichoromethane), and dried with nitrogen gas. . Here, trifluoroacetic acid (TFA), phenol, thioanisole, H ₂ O, and triisopropylsilane were added at a ratio of 85:5:5:2.5:2.5 (v/v), respectively. The mixed solution was added and reacted for 2 to 3 hours to remove the protecting group and separate the peptide from the resin, and then precipitate the peptide with diethylether to obtain the peptide. The obtained crude peptide was purified on a purified reverse phase (RP)-HPLC column (Delta Pak, C18300Å, 15,19.0mm×30 cm, It was purified using Waters, USA). The synthetic peptide was hydrolyzed with 6N hydrochloric acid at 110°C, the residue was concentrated under reduced pressure, dissolved in 0.02N hydrochloric acid, and the amino acid composition was measured using an amino acid analyzer (Hitachi 8500 A), followed by MALDI mass spectrometry to confirm the purity and molecular weight of the peptide. (Hill, et al., Rapid Commun. Mass Spectrometry, 5: 395, 1991) was performed.

As a result, it was confirmed that the peptide described in the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4 in Table 1 was synthesized with a purity of 95% or more, and its molecular weight was the same as the expected molecular weight.

Peptide name amino acid sequence sequence number Molecular Weight PEP27 MRKEFHNVLSSGQLLADKRPARDYNRK-NH ₂ One 3228.7 PEP27-1 MWKWFHNVLSSGQLLADKRPARDYNRK-NH ₂ 2 3316.9 PEP27-2 MWKWFHNVLSWGWLLADKRPARDYNRK-NH ₂ 3 3474.1 PEP27-5 MWKWFHNVLSSGQLLADKWWAWWYNWW-NH ₂ 4 3625.2

Table 2 shows the types of substituted amino acids and positions of substitution in the new peptides (SEQ ID NOs: 2 to 4) based on the sequence of PEP27 (SEQ ID NO: 1).

amino acid number PEP27 PEP27-1 PEP27-2 PEP27-5 One M 2 R W W W 3 K 4 E W W W 5 F 6 H 7 N 8 V 9 L 10 S 11 S W 12 G 13 Q W 14 L 15 L 16 A 17 D 18 K 19 R W 20 P W 21 A 22 R W 23 D W 24 Y 25 N 26 R W 27 K W

[Blank: No amino acid substitution]

2. Measurement of antibacterial activity

In order to evaluate the antibacterial activity of the peptides prepared by the method of 1 above, the Minimum Inhibitory Concentration (MIC) value, which is the minimum concentration of the peptide that does not cause cell division, was measured.

Specifically, the strains listed in Table 3 below were purchased, cultured to mid-log phase in a medium with a composition suitable for each strain, and then diluted to a concentration of 2×10 ⁴ cells/100 ㎕ and micro It was prepared in a titration plate (Nunc, USA). Then, the PEP27, PEP27-1, PEP27-2, or PEP27-5 peptide synthesized in step 1 above was serially diluted 1/2-fold and added to each well, incubated at 37°C for 18 hours, and micro-titrated. The MIC value for each strain was determined by measuring absorbance at a wavelength of 600 nm using a plate reader (Merck Elisa reader, Germany). The parent peptide, PEP27, was used as a control, and buforin 2 and melittin were used as comparison peptides.

As a result, as shown in Table 4 below, the PEP27-2 peptide was confirmed to exhibit significantly superior antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant bacteria compared to the control parent peptide PEP27 and other new peptides.

3. Measurement of hemolytic activity

In order to compare the cytotoxicity of the peptides prepared by the method of 1 above, the red blood cell hemolytic activity of the synthesized peptides was measured.

Specifically, mouse (Balb/c, 6-week-old, male) red blood cells were diluted with PBS (pH 7.0) to a concentration of 8%, and PEP27, PEP27-1, PEP27-2, or PEP27-5 peptides were added at 3.13 and 6.25, respectively. , were treated at concentrations of 12.5, 25.0, 50.0, and 100.0 μM/well, and reacted at 37°C for 1 hour. Then, the amount of hemoglobin contained in the supernatant obtained by centrifugation at 1,000 x g was confirmed by measuring the absorbance at a wavelength of 414 nm. As a control that serves as a standard for the degree of cell destruction, the absorbance of the supernatant obtained after treatment with 1% Triton In percentage terms, the hemolysis activity (hemolysis) of each peptide was calculated using Equation 1 below.

[Equation 1]

Red blood cell destruction ability (%) = (Absorbance A-Absorbance B)/(Absorbance C-Absorbance B) × 100

(In the above equation, absorbance A represents the absorbance of the reaction solution treated with each peptide measured at a wavelength of 414 nm; absorbance B represents the absorbance of the reaction solution treated with PBS measured at a wavelength of 414 nm; absorbance C is Shows the absorbance of the reaction solution treated with 1% Triton X-100 measured at a wavelength of 414 nm.)

As a result, as shown in Table 5 below, it was confirmed that the parent peptide, PEP27 peptide, showed only a 3.58% hemolytic effect on mouse red blood cells when treated at a concentration of 100 μM, whereas the PEP27-2 peptide showed a 75.8% hemolytic effect on red blood cells even at a concentration of 100 μM. It was confirmed that hemolysis was induced and that toxicity was increased. However, this indicates increased toxicity due to increased activity, and it was confirmed that there was no toxicity within the range of antibacterial activity.

Red blood cell destruction ability (%) Peptide concentration
100 μM Peptide concentration
50 μM Peptide concentration
25 μM Peptide concentration
12.5 μM Peptide concentration
6.25 μM Peptide concentration
3.13 μM PEP27
(SEQ ID NO: 1) 3.58 3.64 2.76 1.79 1.26 0 PEP27-1 (SEQ ID NO: 2) 50.65 44.51 25.75 1.97 1.05 0 PEP27-2 (SEQ ID NO: 3) 75.84 62.47 40.51 14.96 2.30 0 PEP27-5 (SEQ ID NO: 4) 3.25 2.34 0 0 0 0 Buforin 2 5.77 3.10 2.05 0.92 0.29 0 Melittin 99.17 97.76 83.99 69.75 49.41 27.33

4. Confirmation of cytotoxicity in normal cell lines

To confirm the cytotoxicity of the peptides prepared by the method of 1 above in normal cell lines, toxicity was measured using a human keratinocyte cell line (HaCaT cell line, Dr. NE. Fusenig, Heidelberg, Germany).

Specifically, HaCaT cells cultured in DMEM medium containing 10% FBS (Fetal Bovine Serum) were distributed at 2 × 10 ⁵ cells/well on a micro titration plate and cultured for 24 hours, and then PEP27, PEP27-1, and PEP27-2 Alternatively, PEP27-5 peptide was treated at a concentration of 3.13, 6.25, 12.5, 25.0, 50.0, or 100.0 μM/well, respectively, and reacted in a 5% CO ₂ incubator for 24 hours. 24 hours later, 100 ㎕ of a reaction solution containing 0.5 mg/ml MTT (Thiazolyl Blue Tetrazolium Bromide) dissolved in phosphate buffered saline (PBS) was added to each well and reacted for 4 hours. Then, the supernatant was removed, 200 μl of DMSO (dimethyl sulfoxide) was added to dissolve the formed MTT crystals, and cell viability was confirmed by checking the wavelength at 560 nm.

As a result, when treated with 100 μM of the parent PEP27 peptide, as shown in Table 6 below, HaCaT cells showed 100% cell viability, confirming that there was no cytotoxicity. On the other hand, the cell viability of PEP27-2 peptide was confirmed to be 69.3% even at a concentration of 100 μM. This confirmed that the cytotoxicity was lower than the hemolysis phenomenon, and it was confirmed that the antibacterial peptide of the present invention had increased antibacterial activity compared to the parent peptide and had no cytotoxicity within the range of antibacterial activity.

Cell viability (%) Peptide concentration
100 μM Peptide concentration
50 μM Peptide concentration
25 μM Peptide concentration
12.5 μM Peptide concentration
6.25 μM Peptide concentration
3.13 μM PEP27
(SEQ ID NO: 1) 100 100 100 100 100 100 PEP27-1 (SEQ ID NO: 2) 42.38 66.45 83.21 100 100 100 PEP27-2 (SEQ ID NO: 3) 30.67 42.24 66.75 99.01 100 100 PEP27-5 (SEQ ID NO: 4) 100 100 100 100 100 100 Buforin 2 100 100 100 100 100 100 Melittin 6.54 11.25 28.21 44.25 78.25 98.21

5. Circular dichroism spectrum measurement

In order to confirm whether the peptide prepared by the method of 1 above induces an α-helical structure, which is a secondary structure, it was measured using the circular dichroism method.

Specifically, 10mM PBS (pH 7.4), PE/PG (L-α-Phosphatidylethanolamine/L-α-Phosphatidyl-DL-glycerol; 7/3, w/w) or PC/CH/SM (L-α-phosphatidylcholine /cholesterol/sphingomyelin); 1/1/1, w/w/w) of PEP27, PEP27-1, PEP27-2, or PEP27-5 peptides at 40 μM in 0.1 μM suspension of large unilamellar vesicles (LUV). After applying it to a cm-length (path-length) cell, the temperature was fixed at 25°C in a jasco 810 spectrophotometer and the circular dichroism spectrum was measured. The α-helical structure calculation formula for the circular dichroism spectrum was used as Equation 2 below.

[Equation 2]

(θ _obs in Equation 2 above represents the millidegrees of the signal, l represents the optical path-length of the cell size (cm), and c represents the concentration of the added peptide (mol/L) indicates)

As a result, when the peptide was added to a 10 mM PBS solution, no structure was formed, whereas when the peptide was added to a LUV solution composed of PE/PG (7/3) or PC/CH/SM (1/1/1), it did not form a structure. Although there were differences in degree, it was confirmed that all peptides formed an α-helical structure, which is a secondary structure. As a result, the antibacterial peptide of the present invention has an α-helical structure in PE/PG (7/3), which is similar to the membrane of microbial bacteria, and PC/CH/SM (1/1/1), which is similar to the membrane of eukaryotic cells. was confirmed to form (Figure 1).

6. Flow cytometry measurement

In order to confirm whether the peptide prepared by method 1 above acts on the bacterial membrane, the PEP27-2 peptide, which has better antibacterial activity than the parent peptide, was analyzed using flow cytometry.

Specifically, the minimum inhibitory concentration (MIC) value of the parent PEP27 peptide or PEP27-2 peptide was treated with E. coli and then reacted at 37°C for 1 hour. Afterwards, the supernatant was removed using a centrifuge (10,000 rpm), and then stained with propidium iodide (PI) at a concentration of 10 μg/ml for 30 minutes at 4°C. Then, unbound propidium iodide was removed using a centrifuge, 1 ml of physiological saline (PBS) was added to remove cell aggregation, and the effect of the peptide on the bacterial membrane was confirmed using a Bechman flow cytometer. did.

As a result, it was confirmed that the membrane destruction ability of PEP27-2 (21.84%) peptide was slightly increased compared to PEP27 (19.9%) in E. coli cells. However, it was confirmed that the above peptides do not have the ability to damage bacterial membranes like Buforin 2, a cell-penetrating peptide. This confirmed that PEP27 and PEP27-2 kill E. coli without destroying the E. coli membrane. Among the control peptides, melittin, which destroys bacterial cell membranes, was confirmed to have a cell membrane-destroying ability against E. coli (91.6%), confirming that it has a different mechanism from melittin.

7. Analysis of the presence or absence of binding between antibacterial peptides and DNA

In order to specifically confirm the mechanism by which the synthetic peptide prepared by method 1 above exhibits antibacterial activity, it was tested whether the PEP27, PEP27-1, PEP27-2 or PEP27-5 synthetic peptide binds to DNA, an internal material of bacteria. This was confirmed through electrophoresis.

Specifically, 300 ng of plasmid DNA (pRSETB) was mixed with peptide in different ratios (peptide/DNA ratios were DNA alone, 0.25:1, 0.5:1, 1:1, 1.5:1, 2:1, 3:1, respectively). After reacting at 37°C for 10 minutes (performed at 4:1 ratio), electrophoresis was performed on a 1% agarose gel, stained with ethidium bromide (EtBr), and confirmed by UV.

As a result, as shown in Figure 3, melittin, a comparative peptide, did not bind to DNA at all, and PEP27, PEP27-2, and Buforin 2, a control peptide, all showed binding to DNA, although there were differences in degree (Figure 3). At this time, it was confirmed that PEP27-2 peptide had higher DNA binding ability than PEP27. Through the above results, it was confirmed that the PEP27-2 peptide exhibits higher antibacterial activity due to the substitution of some amino acid residues due to the higher binding ability of the PEP27-2 peptide to DNA, an internal material of bacteria, compared to the parent peptide, PEP27.

8. Scanning Electron Microscope (SEM) analysis

In order to confirm whether the synthetic peptide prepared by method 1 above damages the bacterial cell membrane, it was confirmed using a scanning electron microscope.

Specifically, E. coli and Staphylococcus aureus cells were suspended in PBS at a concentration of 0.2 at OD ₆₀₀ , then treated with PEP27-2 peptide at the minimum inhibitory concentration (MIC) and reacted at 37°C for 1 hour. As a control group, a strain without peptide was used. After culturing, the cells were recovered, treated with 2.5% glutaraldehyde at 4°C for 18 hours, and then washed twice with PBS buffer solution. The fixed cells were sequentially dehydrated using 100% ethanol and diluted ethanol (50%, 70%, 90%, and 100%) for 10 minutes, then the specimen was dried, coated with platinum, and subjected to low vacuum scanning electron microscopy. It was confirmed using .

As a result, it was confirmed that while the untreated control cells as shown in Figure 4 had a bright and smooth surface, the cells treated with melittin, a membrane-destroying peptide used as a comparative control, had significant membrane damage. The cell surface exposed to the peptide was observed to have blister-like damage with holes forming, and in some cases, leakage of cytoplasmic contents was observed. However, cells treated with PEP27-2 peptide showed almost no cell damage, almost the same as the control group. It was confirmed that this kills bacteria without destroying the cell membrane.

9. Cellular uptake of peptides

The ability of the synthetic peptide prepared by method 1 above to penetrate cells was confirmed through phase contrast microscopy. Specifically, human keratinocyte cell line (HaCaT cells) was dispensed at ² Orecein isothiocyanate (FITC)-labeled PEP27-2 (FITC-PEP27-2) was treated at a concentration of 5 μM and cultured in a 5% CO ₂ incubator for 1 hour. After 1 hour of reaction, the nuclei of the cells were stained with Hoechst 33342 nucleic acid stain (Invitrogen, Carlsbad, CA, USA; 8 μM) for 10 minutes. After washing with PBS three times, intracellular fluorescence was imaged using a phase contrast microscope (EVOS™ FL Auto 2 Imaging System, Thermo Fisher Scientific).

As a result, as shown in Figure 5, FITC-PEP27-2 was clearly absorbed by HaCaT cells and evenly distributed with intracellular green fluorescence, appearing in both the cytoplasm and nucleus. To confirm the nuclear localization of PEP27-2, the nuclei of cells treated with FITC-labeled peptide were counterstained with the nuclear stain Hoechst 33342, and it was confirmed that PEP27-2 was distributed in both the cytoplasm and nucleus of HaCaT cells. In HaCaT cells, FITC-PEP27-2 was confirmed to be absorbed into the cytoplasm and nucleus. The control peptide, TAT (GRKKRRQRRRPQ), is a highly positively charged cell-penetrating peptide with 11 amino acid residues and, like the PEP27-2 peptide, was confirmed to be absorbed into the cytoplasm and nucleus in HaCaT cells.

10. Measurement of antibiofilm activity

The biofilm inhibition concentration value of PEP27-2, the peptide with the best antibacterial activity among the peptides prepared by method 1 above, was measured.

Specifically, among the strains listed in Table 3, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were cultured in each medium to mid-log phase, and then cultured at 5×10 ⁴ cells/100. The cells were diluted to a concentration of ㎕ and inoculated into a microplate (SPL). Then, 10 ㎕ of Pse-T2 peptide diluted 1/10 times with 10 mM PBS solution (pH 7.2) was added to each well and incubated at 37°C for 24 hours. After completely removing the supernatant, it was fixed with 100% methanol for 15 minutes, stained with crystal violet staining solution for 1 hour, washed three times, dissolved in 95% ethanol, and measured for absorbance at a wavelength of 595 nm using a micro titration plate reader. was measured to confirm the minimum biofilm inhibitory concentration value for each strain.

As a result, as shown in Table 7 below, it was confirmed that in all strains, the PEP27-2 peptide exhibited strong biofilm inhibitory activity almost similar to that of the control peptide, melittin.

11. Confirmation of antibacterial activity of antibacterial peptides and antibiotics

In order to compare the antibacterial activity of PEP27-2, which is the peptide with the best antibacterial activity among the peptides prepared by the method of 1 above, with existing antibiotics, the same Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aerosi) were used. The MIC concentration of the antibiotic for the Nosa) strain was confirmed. Specifically, the target strain was prepared in the same manner as in Example 2, and treated with the antibiotics Meropenem, Ceftazidime, Ceftriaxone, or Vancomycin, respectively, to determine the MIC of the antibiotic. was confirmed.

As a result, as shown in Table 8 below, the MIC of PEP27-2 for 11 strains was confirmed to be in the range of 2 to 4 μM. The MICs of Meropenem, Ceftazidime, Ceftriaxone, and Vancomycin were confirmed to exhibit antibacterial activity in the range of 0.25 to 512 μM (Table 8).

12. Confirmation of synergistic effect when combined treatment with antibacterial peptides and antibiotics

Using the MIC values determined for each mixture, the synergistic effect of peptides and antibiotics in antibacterial activity against strains was evaluated. For evaluation, fractional inhibitory concentration (FIC) analysis was performed using a checkerboard assay.

Specifically, 100 ㎕ of bacteria ( ⁵ were added one at a time. Afterwards, the antibiotic solution was diluted and 50 μl was added to each well, and the same procedure was performed under different conditions to determine the MIC value of each mixed solution. The FIC value was calculated as follows: FIC index = FIC (A) + FIC (B) = [A]/MIC (A) + [B]/MIC (B), where [A] is the PEP27-2 antibiotic. If present, MIC(A) is the MIC of PEP27-2 alone and FIC(A) is the FIC of PEP27-2. [B], MIC (B) and FIC (B) are values corresponding to antibiotics. According to the calculated FIC value, the following evaluations were made: <0.5, Synergy effect; 0.5~4, Indifference; >4.0, Antagonism.

As a result, it was confirmed that no combination of peptide and antibiotic had an antagonistic effect. As a result, as shown in Table 9 below, a synergistic effect was confirmed for meropenem, ceftazidime or ceftriaxone in combination with 0.25x MIC of PEP27-2 in Staphylococcus aureus CCARM 3089 and CCARM 3090. In addition, in the case of Pseudomonas aeroginosa 4891 and Staphylococcus aureus MW2, a synergistic effect was confirmed only with meropenem or ceftriaxone, respectively, when combined with 0.25x MIC of PEP27-2 (Tables 9 and 10). As shown in Tables 9 and 10 below, synergistic activity was confirmed in most antibiotic-resistant strains compared to a small number of standard strains.

13. Measurement of healing ability of abscesses infected with multidrug-resistant Staphylococcus aureus

The efficacy of Pse-T2, the peptide with the best antibacterial activity among the peptides prepared by the method of 1 above, was evaluated in vivo using a mouse model.

Specifically, Staphylococcus aureus MW2 ( S. aureus MW2-GFP, 1×10 ⁹ CFU/100㎕ PBS) with the GFP gene inserted into the epidermis of the back (dorsal) of 6-7 week old BALB/c mice. infected. 2 hours after infection, PEP27-2 (0.2 mg/kg, 50 ㎕) alone, ceftriaxone (0.2 mg/kg, 50 ㎕) alone, or a combination of peptide and antibiotics (0.05 mg/kg PEP27-2 +0.1 mg/ kg ceftriaxone, 50 μl) was injected. Control mice were injected with the same amount of PBS (20 μl) without peptide. At 1, 2, 3, 4, 5, and 7 days after treatment, the morphology of the abscess was photographed and the abscess and surrounding tissue were recovered. Abscess tissue was homogenized in 500 μl of sterilized PBS using a tissue grinder. Serial dilutions of the homogenate were plated on agar plates and cultured for 24 hours to quantify Staphylococcus aureus MW2 strain.

As a result, it was confirmed that no abscess was formed in the experimental group injected only with PBS, which was not infected with Staphylococcus aureus MW2 strain (FIG. 6). On the other hand, in the experimental group where only PBS was injected after infection with Staphylococcus aureus MW2, abscesses formed after 1 day, and the size of the abscess did not decrease until the 7th day, and it was found that the abscess was severely inflamed. However, after infection with Staphylococcus aureus MW2, the experimental group injected with only PEP27-2, the experimental group injected only with ceftriaxone, and the experimental group injected with only PEP27-2 + ceftriaxone had a higher incidence of abscess compared to the experimental group injected with PBS. It was found that the size was small. On the other hand, it was visually confirmed that the abscess healing effect when treated in combination with PEP27-2 was significantly similar or slightly greater than when treated with PEP27-2 alone after infection with Staphylococcus aureus MW2 (FIG. 6). To more clearly confirm the abscess healing effect, the homogenate of the abscess tissue was cultured to determine the bacterial count. After bacterial infection and treatment with PEP27-2 or ceftriaxone, the bacterial count was 3 compared to 0 day, respectively. It decreased by 80.0% or 46.0% at 1 day and 89.3% or 61.5% at 7 days. The number of colonies after combined treatment was 91.1% at 7 days, confirming that the number of Staphylococcus aureus MW2 bacteria was lower than after single treatment (FIG. 7). This showed that the combination treatment had a synergistic effect in improving the removal of bacteria from the site of infection compared to single treatment with PEP27-2 or ceftriaxone. In addition, it was confirmed that treatment with PEP27-2 alone was effective in removing strains from the abscess area.

14. Live fluorescence imaging of an abscess mouse model

Among the peptides prepared by method 1 above, fluorescent (GFP)-labeled Staphylococcus aureus MW2 (Staphylococcus aureus MW2-GFP), which has the highest antibacterial activity, was used to track the progress of infection in real time. . Fluorescence images were taken for 6 hours, 1 day, 2 days, and 3 days from the start of infection using a FOBI fluorescence imaging system (Neo Science, Suwon, Korea) and analyzed with live image software.

As a result, there was no difference in fluorescence signal between groups at 0 h after infection with Staphylococcus aureus MW2-GFP at a high bacterial dose (1 × ¹⁰ CFU) (Figure 8). Fluorescent signals continued to increase for 3 days on the skin surface over abscesses treated only with Staphylococcus aureus MW2-GFP. The fluorescence signal after PEP27-2 treatment alone was similar to the combination treatment, but the fluorescence signal of ceftriaxone alone did not decrease (Figure 8). Based on the fluorescent abscess area, it was confirmed that the combined treatment of PEP27-2 and ceftriaxone showed a synergistic effect.

15. Staining of abscess tissue sections infected with multidrug-resistant Staphylococcus aureus

Among the abscess area and inflammation area tissues recovered in 13 above, the 7th day tissue was fixed in 4% buffered formalin. Inflammatory areas or abscesses fixed with formalin were embedded in paraffin, and the cross-sections were stained with hematoxylin and eosin.

As a result, the infection area of mice infected with Staphylococcus aureus MW2 alone expanded from skin tissue to deep skeletal muscle tissue, and neutrophils and macrophages were observed around the infection area. Deep inflammation of the upper dermis, skeletal muscle damage and necrosis resulted in the formation of a large abscess with crust/crust on the upper surface (Figure 9). Bacterial counting and live fluorescence imaging confirmed that the scabs visible on the skin surface contained abundant Staphylococcus aureus MW2. It was confirmed that the number of abscess areas, visible scabs formed on the skin surface, and number of bacteria were significantly reduced in mice treated with PEP27-2, ceftriaxone, or their combination 2 hours after infection with Staphylococcus aureus MW2. . However, it was confirmed in tissue sections that the inflammatory reaction occurred most significantly during the combined treatment (FIG. 9).

16. Pro-inflammatory cytokine gene expression analysis using quantitative real-time PCR

Tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) The mRNA expression of the same inflammatory mediators changes during skin infection and wound healing. The expression of inflammatory genes was measured in mice that were not infected with bacteria, mice that were infected, mice that were treated with PEP27-2 alone, ceftriaxone alone, or in combination after infection, and the difference between PEP27-2 treatment alone and in combination with antibiotics was measured. Efficacy was verified.

Specifically, total RNA was isolated from wound tissue using Trizol reagent. The prepared total RNA was synthesized into cDNA using a cDNA synthesis kit. qPCR was performed using qPCR 2x premix (SYBR Green), and the expression levels of inflammatory genes TNF-α, IL-1β, IL-6, iNOS, and COX-2 were confirmed. The expression level was quantified based on β-actin, and the PCR process was 50°C for 2 minutes, 95°C for 10 minutes; 95°C for 15 seconds, 60°C for 1 minute, 40 cycles; 15 seconds at 95°C, 1 minute at 60°C, 30 seconds at 95°C, and 15 seconds at 60°C. Gene expression was calculated after normalizing to β-actin levels using the ΔΔCt method. The transcription of control cells was set to 1 and the other experimental groups were multiples of this value.

As a result of confirming the expression of TNF-α, IL-1β, IL-6, iNOS, and COX-2, it was observed that the expression of each of the above inflammatory cytokines was increased in the abscess area of the skin infected with Staphylococcus aureus MW2 strain, It was confirmed that the expression of inflammatory cytokines was suppressed under conditions in which skin wounds infected with bacteria were treated with PEP27-2 alone, ceftriaxone alone, or in combination (FIG. 10). Combination treatment with PEP27-2 and ceftriaxone showed greater inhibition of inflammatory cytokines except TNF-α than treatment with either agent alone. For TNF-α expression, treatment with PEP27-2 alone and combination of PEP27-2 and ceftriaxone were similarly inhibited. This confirmed that the combination treatment had a synergistic effect in suppressing the expression of some inflammatory mediators induced in mice infected with Staphylococcus aureus MW2. Therefore, it was confirmed that the combined use of PEP27-2 and ceftriaxone injection exerts an antibacterial effect against Staphylococcus aureus MW2 in vivo and suppresses inflammation that occurs in response to infection.

From the above experimental results, the three PEP27 analogues of the present invention (SEQ ID NO: 2 to SEQ ID NO: 4) have low cytotoxicity to normal cells, and are superior to or similar to the parent peptide, PEP27, against Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant bacteria. It was confirmed that it exhibits antibacterial activity against. In particular, among the synthesized peptide analogs, PEP27-2 peptide (SEQ ID NO: 3) was confirmed to exhibit significantly superior antibacterial activity compared to the parent peptide, PEP27. In addition, it was confirmed that the combined treatment of the synthesized peptide analog and antibiotics showed synergistic antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant bacteria.

Preparation Example 1: Preparation of pharmaceutical preparation

1-1. manufacture of powders

ingredient Weight (mg) Peptide of the present invention 20mg lactose 20mg

After mixing the above ingredients, they were filled into an airtight bubble to prepare a powder.

1-2. manufacture of tablets

ingredient Weight (mg) Peptide of the present invention 10 mg corn starch 100mg lactose 100mg Magnesium Stearate 2mg

After mixing the above ingredients, tablets were manufactured by tableting according to a conventional tablet manufacturing method.

1-3. Manufacturing of capsules

ingredient Weight (mg) Peptide of the present invention 10 mg crystalline cellulose 3mg lactose 14.8mg Magnesium stearate 0.2mg

After mixing the above ingredients, a capsule was prepared by filling a gelatin capsule according to a typical capsule manufacturing method.

1-4. Manufacture of liquid

ingredient weight Peptide of the present invention 20mg Iseonghwadang 10g Mannatol 5 g Purified water Appropriate amount

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, adjust the total to 100 ml by adding purified water, and fill it in a brown bottle. The solution was prepared by sterilization.

1-5. Manufacturing of injectable drugs

ingredient Added amount Peptide of the present invention 10mg/ml Dilute hydrochloric acid BP until pH reaches 7.6 Sodium chloride BP for main use Up to 1 ml

The peptide of the present invention was dissolved in an appropriate volume of sodium chloride BP for main use, the pH of the resulting solution was adjusted to pH 7.6 using dilute hydrochloric acid BP, and the volume was adjusted using sodium chloride BP for main use and thoroughly mixed. The solution was filled into a 5 ml Type I ampoule made of transparent glass, sealed under an upper grid of air by dissolving the glass, and sterilized in an autoclave at 120°C for more than 15 minutes to prepare an injection solution.

Preparation Example 2: Preparation of cosmetics

2-1. Soft lotion (skin)

To prepare an antibacterial softening lotion containing the peptide of the present invention, it can be prepared according to a conventional manufacturing method in the cosmetics field by mixing as shown in Table 16 below.

ingredient Content (% by weight) Peptide of the present invention 0.1~30 1,3-butylene glycol 3.0 glycerin 5.0 Polyoxyethylene (60) hydrogenated castor oil 0.2 ethanol 8.0 citric acid 0.02 Sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water To 100

2-2. Nutritional lotion (lotion)

To prepare an antibacterial nutritional lotion containing the peptide of the present invention, it can be prepared according to a typical manufacturing method in the cosmetics field by mixing as shown in Table 17 below.

ingredient Content (% by weight) Peptide of the present invention 0.1~30 squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 Yuchang Tree Oil 0.1~30 1,3-butylene glycol 8.0 glycerin 5.0 Polyoxyethylene (60) hydrogenated castor oil 0.2 ethanol 8.0 citric acid 0.02 sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water To 100

2-3. essence

To prepare an antibacterial essence containing the peptide of the present invention, it can be prepared according to a conventional manufacturing method in the cosmetics field by mixing as shown in Table 18 below.

ingredient Content (% by weight) Peptide of the present invention 0.1~30 sitosterol 1.7 Polyglyceryl 2-oleate 1.5 ceramide 0.7 Ceteares-4 1.2 cholesterol 1.5 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Carboxy vinyl polymer 0.2 xanthan gum 0.2 antiseptic a very small amount Spices a very small amount Purified water To 100

2-4. Face wash (cleansing foam)

To produce an antibacterial face wash (cleansing foam) containing the peptide of the present invention, it can be prepared according to a typical manufacturing method in the cosmetics field by mixing as shown in Table 19 below.

ingredient Content (% by weight) Peptide of the present invention 0.1~30 Sodium N-acylglutamate 20.0 glycerin 10.0 PEG-400 15.0 propylene glycol 10.0 POE(15) oleyl alcohol ether 3.0 Laurin derivative 2.0 Methylparaben 0.2 EDTA-4Na 0.03 Spices 0.2 Purified water To 100

2-5. Nutrition cream

To prepare an antibacterial nutritional cream containing the peptide of the present invention, it can be prepared according to a conventional manufacturing method in the cosmetics field by mixing as shown in Table 20 below.

ingredient Content (% by weight) Peptide of the present invention 0.1~30 vaseline 7.0 liquid paraffin 10.0 beeswax 2.0 Polysorbate 60 2.5 Sorbitan sesquioleate 1.5 squalane 3.0 propylene glycol 6.0 glycerin 4.0 Triethanolamine 0.5 xanthan gum 0.5 Tocophenylacetate 0.1 Fragrances, preservatives a very small amount Purified water To 100

2-6. massage cream

To prepare an antibacterial massage cream containing the peptide of the present invention, it can be prepared according to a conventional manufacturing method in the cosmetics field by mixing as shown in Table 21 below.

ingredient Content (% by weight) Peptide of the present invention 0.1~30 propylene glycol 6.0 glycerin 4.0 Triethanolamine 0.5 beeswax 2.0 Tocophenylacetate 0.1 Polysorbate 60 3.0 Sorbitan sesquioleate 2.5 Cetearyl alcohol 2.0 liquid paraffin 30.0 xanthan gum 0.5 Fragrances, preservatives a very small amount Purified water To 100

2-7. pack

To prepare an antibacterial pack containing the peptide of the present invention, it can be prepared according to a conventional manufacturing method in the cosmetics field by mixing as shown in Table 22 below.

ingredient Content (% by weight) Peptide of the present invention 0.1~30 propylene glycol 2.0 glycerin 4.0 polyvinyl alcohol 10.0 ethanol 7.0 PG-40 Hydrogenated Castor Oil 0.8 Triethanolamine 0.3 Fragrances, preservatives a very small amount Purified water To 100

<110> Industry-Academic Cooperation Foundation, Chosun University <120> Novel peptide derived from PEP27 peptide and uses it <130> 20P12021 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 27 <212> PRT <213> Artificial Sequence <220> <223>PEP27 <220> <221> MOD_RES <222> (27) <223> AMIDATION, <400> 1 Met Arg Lys Glu Phe His Asn Val Leu Ser Ser Gly Gln Leu Leu Ala 1 5 10 15 Asp Lys Arg Pro Ala Arg Asp Tyr Asn Arg Lys 20 25 <210> 2 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> PEP27-1 <220> <221> MOD_RES <222> (27) <223> AMIDATION, <400> 2 Met Trp Lys Trp Phe His Asn Val Leu Ser Ser Gly Gln Leu Leu Ala 1 5 10 15 Asp Lys Arg Pro Ala Arg Asp Tyr Asn Arg Lys 20 25 <210> 3 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> PEP27-2 <220> <221> MOD_RES <222> (27) <223> AMIDATION, <400> 3 Met Trp Lys Trp Phe His Asn Val Leu Ser Trp Gly Trp Leu Leu Ala 1 5 10 15 Asp Lys Arg Pro Ala Arg Asp Tyr Asn Arg Lys 20 25 <210> 4 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> PEP27-5 <220> <221> MOD_RES <222> (27) <223> AMIDATION, <400> 4 Met Trp Lys Trp Phe His Asn Val Leu Ser Ser Gly Gln Leu Leu Ala 1 5 10 15 Asp Lys Trp Trp Ala Trp Trp Tyr Asn Trp Trp 20 25

Claims (9)

An antibacterial composition against methicillin-resistant Staphylococcus aureus , comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 2 or 3.
The antibacterial composition according to claim 1, further comprising at least one antibiotic selected from the group consisting of meropenem, ceftazidime, and ceftriaxone.
The antibacterial composition according to claim 1, wherein the methicillin-resistant Staphylococcus aureus is S. aureus MW2.
A pharmaceutical composition for preventing or treating skin abscesses, comprising the antibacterial composition of any one of claims 1 to 3.
A cosmetic composition comprising the antibacterial composition of any one of claims 1 to 3.
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